In contrast to solid-phase immobilised miniaturised nucleic acid libraries, about which there are a large number of publications in technical journals and patents (S. P. A. Fodor et al. (1991), Light-directed, spatially addressable parallel chemical synthesis. science 251.767-773; E. M. Southern et al. (1992), Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models. Genomics 13:1008-1017; G. McGall. et al. (1996), Light-directed synthesis of high-density oligonucleotide arrays using semiconductor photoresists, Proc. Natl. Acad. Sci. USA 26:13S55-13560, M. Chee et al. (1996), Accessing genetic information with high density DNA arrays. Science 274:610-614; 5. Singh-Gasson et al. (1999), Maskiess fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nat. Biotechnol. 17:974-978; S. P. A. Fodor et al. (1995), Arrays of materials attached to a substrate. U.S. Pat. No. 5,744,305; S. P. A. Fodor et at. (1995), Very large scale immobilised polymer synthesis. U.S. Pat. No. 5,424,186; G. H. McGall et al. (1995), Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces. U.S. Pat. No. 5,412,087, A. S. Heuermann (1999), Method and device for photolithographic production of DNA, PNA and protein chips. WO 9960156A2, G. H. McGall und N. Q. Nam (2000), Synthesis of oligonucleotide arrays using photocleavableprotecting groups. U.S. Pat. No. 6,022,963) solid-phase immobilised greatly miniaturised peptide or peptidomimetic libraries have hitherto been described only in a small number of works (S. P. A. Fodor et al. (1991), light-directed, spatially addressable parallel chemical synthesis. Science 25 1:767-773; C. P. Holmes et al. (1995), The use of light-directed combinatorial peptide synthesis in epitope mapping. Biopolymers 37:199-211; M. C. Pirrung et al. (1992), Large scale photolithographic solid phase synthesis of polypeptides and receptor binding thereof U.S. Pat. No. 5,143,854, M. C. Pirrung et al. (1995), Large scale photolithographic solid phase synthesis of an array of polymers. U.S. Pat. No. 5,405,783).
One of the main reasons why miniaturised peptide libraries, so-called ‘peptide chips’, have hitherto not yet achieved widespread dissemination, is the relatively high level of expenditure involved in the production of such arrangements if the conventional photolithographic synthesis methods used in relation to ‘DNA chips’ on planar supports are used. In the case of nucleic acids, those methods make use of the fact that there are only four different naturally occurring nucleotides (deoxyadenosine, deoxycytidine, deoxyguanosine and thymidine) for producing the deoxyribonucleic acid oligomers, that the coupling times when forming the oligonucleotides are relatively short (less than 30 minutes) and the yields of the individual coupling steps are very good (more than 99%). All three criteria have a crucial influence on the production time for such chips and thus the economy of the entire process.
In the photolithographic synthesis of oligonucleotides, firstly a support which is completely protected with a photolabile protective group is cleared of protection in positionally directed fashion by irradiation with light at the locations at which for example a thymidine phosphate is to be applied, and then the entire support is incubated with thymidinephosphoamidite which is photolabily protected at the 5′-OH and suitable coupling reagents. Coupling is thus effected only at the desired locations and the entire procedure has to be repeated for the other three nucleotides before the first dinucleotide can be synthesised. In the case of a chip which is occupied with 20-mer oligonucleotides 80 coupling, washing and protection-removal cycles are therefore required.
In the case of a peptide however the number of components which can be used for coupling is markedly higher than in the case of the nucleic acids. There are 20 proteinogenic amino acids, some naturally occurring non-proteinogenic amino acids (for example ornithine), the same number of corresponding D-amino acids and a continuously rising number of artificial amino acids such as for example cyclohexylalanine, aminoisobutyric acid, Norvaline, etc, also each in D-and L-form. In summary it can be assumed that at the present time about 100 different amino acids are available for the chemical synthethis of peptides and peptidomimetics. If—considered conservatively—only half of those reagents are used for the synthethis of a substance library, that gives 1000 coupling and protection-removal cycles in the synthesis of a 20-mer peptide or peptidomimetic library. In addition solid-phase peptide synthesis generally affords coupling yields of 85-90% with reaction times of about 30 minutes so that usually at least one repetition of the coupling step with fresh reagents is necessary to achieve the required synthesis yields. This means that extremely long synthesis times of weeks up to several months have to be calculated into the procedure for the production of a peptide or peptidomimetic library immobilised on a two-dimensional support if operation is implemented using photolithographic methods. Consequently hitherto only photolithographic syntheses of short peptides of low sequence variability have been described (S P A Fodor et al (1991) Light-directed, spatially addressable parallel chemical synthesis, Science 251; 767-773, C P Holmes et al (1995), The use of light directed combinatorial peptide synthesis in epitope mapping. biopolymers 37:199-211; M C Pirrung et al (1992), Large scale photolithographic solid phase synthesis of polypeptides and receptor binding thereof U.S. Pat. No. 5,143,854).
An alternative method which considerably reduces the number of working steps is based on the simultaneous protection removal of all support-bonded reaction partners followed by parallel or sequential positionally directed application of the different amino acid reaction mixtures into defined sample areas. In that way it is also possible to synthesise libraries from longer and complex peptides or peptidomimetics in an acceptable time frame. The main problem in this respect however is the cross-contamination which is to be expected of the reactants if the sample areas are close together. Dense packing of the sample areas in turn is desirable in order sufficiently to miniaturise the substance library.
One possible way of resolving the problem involves increasing the surface tension in the regions between the sample areas of the planar support so that the reaction mixtures remain in the form of small droplets in the region of the sample areas. To achieve that aim the support surface must be fluoroalkylated between the sample areas, as described in T M Brennan (1995) Method and apparatus for conducting an array of chemical reactions on a support surface, U.S. Pat. No. 5,474,796; T M Brennan (1997), Method and apparatus for conducting an array of chemical reactions on a support surface, EP 703 825B1 and T M Brennan (1999), Method and apparatus for conducting an array of chemical reactions on a support surface, U.S. Pat. No. 5,985,551. The method however suffers from the disadvantage that without a protective enclosure the drops are severely subjected to evaporation, which can represent a major problem particularly when dealing with very small sample volumes.
An alternative way of resolving the cross-contamination problem lies in the use of a porous membrane which immediately sucks up the amino acid reaction mixture at the location of application (R Frank (1992), Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support, Tetra hedron 48:92 17-9232; R Frank and S Güler (1992), Verfahren zur schnellen Synthese von trägergebundenen oder freien Peptiden oder Oligonukleotiden, damit hergestelltes Flachmaterial, Verwendung sowie Vorrichtung zur Durchführung des Verfahrens, [Method of fast synthesis of support-bonded or free peptides or oligonucleotides, flat material produced therewith, use and apparatus for carrying out the method], WO 92/04366; J Schneider-Mergener (1994), Verfahren zur Synthese und Selektionierung von Sequenzen aus kovalent verbundenen Bausteinen, [Method of synthesising and selecting sequences of covalently joined components], WO 94/20521). The unordered capillaries in the porous membrane, with increasing miniaturisation of the sample areas, result however in cross-contamination and the relatively large surface area of the membrane also results in this method in evaporation of the solvent and thus under some circumstances in an adverse influence on the coupling reaction. A further problem which occurs when using the cellulose membranes usually employed here is in part very great heterogeneity of the synthesised product. In a mass-spectroscopic investigation of the entire material detached from a sample area, both amino-terminally and also carboxy-terminally shortened peptides are found, which occur due to the fact that the synthesis breaks down too early for steric reasons within the membrane and chain re-starts can still take place by virtue of esterification of amino acids directly at the cellulose substrate even in later synthesis cycles. In particular peptides which have been carboxy-terminally shortened by 1-4 amino acids are demonstrated (D Goehmann and A Frey, unpublished results). Complete blocking of the hydroxyl groups of the cellulose by acetylation or similar measures for preventing chain re-starts is prohibited as in that way the support achieves a higher degree of solubility in the solvents usually employed for solid-phase peptide synthesis and loses a considerable amount of mechanical stability (D Goehmann and A Frey, unpublished results). A further disadvantage in the use of membranes lies in their optical properties. In particular cellulose membranes have strong inherent fluorescence in the emission range of many commercially available fluorophores so that the sensitivity of identification of active substances which bind to the target molecules is greatly reduced (J Helfmann, unpublished results).
A further approach for resolving the problem of cross-contamination in charging a planar support with a substance library involves using a microreactor system in which the planar support represents a vessel wall for a plurality of reaction cavities and at the same time the support medium for the target molecules. The reaction cavities which are enclosed on all sides in that way are specifically supplied with the desired reagents and washing solutions for example by changing the positioning of the microreactor block or by means of micropassages, electro-osmotic pumps and microvalves in the reactor block. Waste products are removed in a similar manner (J L Winkler et al (1995), Very large scale immobilised polymer synthesis using 10 mechanically directed flow paths, U.S. Pat. No. 5,384,261; P J Zanzucchi et al (1997), Method of synthesis of a plurality of compounds in parallel using a partitioned solid support, U.S. Pat. No. 5,643,738; P J Zanzucchi et al (1998), Partitioned microelectronic device array, U.S. Pat. No. 5,755,942; S C Cherukuri et al (1999), Method and system for inhibiting cross-contamination in fluids of combinatorial chemistry device, U.S. Pat. No. 5,980,704). A crucial disadvantage of such arrangements however is their susceptibility to particulate impurities in the reaction solutions, in particular if the passages are only a few micrometers in diameter and the direction of flow of the reagents and waste products in the microreactor block is changed a plurality of times. The risk of blockage of the microreactor system is very great in particular in the case of coupling reactions with carbodiimides as the organic urea derivatives which are produced in this case have a strong tendency to crystallisation. In the worst case then the complete microreactor block has to be replaced.
The existing methods and methods described in the literature or patents all have serious disadvantages. Either there is not a large surface area and thus not a high level of detection sensitivity with small external dimensions, or the synthesis times are overall excessively long. Optical detection is severely interfered with due to inherent fluorescence and chain breakages result in a non-homogenous sample. When dense packing of the substance library is involved cross-contamination between various sample areas is to be observed. When dealing with small volumes evaporation influences the results. Handling is complicated or very sensitive in relation to particles.